Project Summary: Maintaining the appropriate balance of excitation (E) vs. inhibition (I) in the nervous system is essential for normal brain function, as disruptions in E/I balance are associated with disorders such as autism spectrum disorder (ASD), schizophrenia, and seizures. Development of therapeutics for ASD and schizophrenia has lagged due to their multifactorial etiology and challenges in modeling relevant biology in scalable in vitro assays. The Optopatch platform recently developed at Q-State Biosciences, which uses engineered optogenetic proteins, custom microscopes, and software, makes it possible to simultaneously stimulate (blue light) and record (red light) electrical activity from ~100 neurons with 1 millisecond temporal resolution, single-cell spatial resolution and high signal-to-noise ratio. In addition to measurements of intrinsic excitability, patterned blue light can be used to probe synaptic connections by stimulating a subset of neurons and recording postsynaptic potentials (PSPs) in all remaining cells. The Optopatch assays, which record signals in individual neurons, can be paired with fluorescent labels of inhibitory neurons to identify compounds that differentially affect signaling in excitatory and inhibitory cells and would be expected to shift the E/I balance. In Phase I of this project, we propose to 1) adapt the established Q-State assays for intrinsic excitability and synaptic transmission to measure the E/I balance and 2) validate these assays using tool compounds and gene knockdowns expected to shift the E/I balance. In Phase II, we propose to 3) screen annotated compound libraries, 4) confirm that proteins targeted by compounds in the library can modulate the E/I balance, and 5) validate that identified targets can also shift the E/I balance in human stem-cell derived neurons and in rodent brain slice. The identified protein targets will serve as a starting point for drug discovery, and identified FDA- approved compounds may be repurposed for new indications. The suite of tools developed here will pave a new path for developing E/I-modulating therapeutics for treating complex neurological disorders such as autism and schizophrenia.